The use of photometric measurements for clinical chemistry, immunology and microbiology are well known. In a horizontal photometer, a light beam is passed through an absorbing solution placed in a cuvette or other vessel with the beam entering the cuvette from one side, passing through the solution, and leaving the cuvette from the other side. The light beam enters the cuvette or other vessel at right angles to the vertical axis of the vessel. The horizontal measurement has been used in colorimetric and turbidity assays.
A vertical measurement may be accomplished using a vertical photometer wherein a vertical light path enters the cuvette from its bottom and passes through the solution to leave the cuvette from the surface of the solution or vice versa. With the vertical measurement, if the sample remains homogeneous and no evaporation of the solution occurs, the vertical and horizontal measurements are equivalent. As actual circumstances in the practical situation results in evaporation and inhomogeneity in the solution causing errors in the horizontal measurement which are not incurred with the vertical measurement technique. The vertical photometer may include a strip reader wherein a strip of microtiter wells will be consecutively read using a transport mechanism. Recently, the clinical assays used in the immunology and microbiology, have utilized enzyme immuno assays (EIA) which have been developed for many diseases including the AIDS HIV virus rather than the previously utilized agglutination assays. The EIA assays are more practically performed using microtiter volumes wherein the vertical photometer is especially useful. Thus, the use of microtiter wells and vertical photometers has grown significantly as even general chemistry applications are utilizing microwell strip or plate reading in order to use less reagent and thus reduce the cost per test.
The use of vertical photometers in clinical assays is therefore becoming quite important. These vertical photometers are calibrated and the calibrations verified by the manufacturer. However, it is both necessary and good practice to routinely monitor the performance of these instruments in the course of clinical practice and may in fact be required by regulatory agencies overseeing such practice.
Currently, a vertical photometer user is limited in the testing and verification methods available. The only commercially available methods involve reading through filters manufactured of glass or other materials. The filters used are usually neutral density filters which do not detect any chromophores or the concentration thereof as will be determined in clinical assay use of the photometer. The filters merely block a predetermined amount of light and pass a particular wavelength of light. The ability of the photometer to accurately detect light can therefore be tested with the use of neutral density filters, but the ability of the instrument to filter light or to detect light of a particular wavelength is not tested.
These techniques are further unrealistic as the photometric instrument is utilized to measure absolute absorbance of different fluids. The use of glass or other filters therefore does not account for several variables which influence the final reading, under normal use of the instrument, such as: the chromophore of the fluid, the lens effect of the vessel in which the fluid is placed, meniscus effects of the liquid within the vessel, fluid effects or movement of a fluid in the vessel and other variables. Failure to account for or regulate these variables undoubtedly contributes to the variations observed and inaccurate results achieved by the instrument users presently. Remarkably, these variations are seen from one instrument manufacturer to another and even among different photometer models from the same manufacturer, even though all of these instruments are referenced to the same standard reference filters and substances provided by the National Bureau of Standards. It has also been found that the use of neutral density filters to check the linearity of a photometer will not yield any indication of whether the photometer will detect the chromophore.
The alternative to the currently available commercial methods of verifying calibrations is to attempt to utilize liquid dyes to simulate actual use in the laboratory. However, there are many problems with this method. Performing such a test is time-consuming, somewhat complex and allows for the introduction of many variables and errors.
Such a laboratory test to determine the accuracy of vertical photometer readings, would involve the use of the simple and well known Beers/Lambert Law which is stated as: EQU A=log (I.sub.o /I)=cl
where A is the absorbance, I.sub.o is the intensity of incident energy, I is the intensity of emergent energy, c is the concentration, l is the thickness of the absorber or path length, and is the molar absorbtivity constant for concentration in moles/liter. However, while applicable in theory, this law ignores the effects of surface tension by assuming that the liquid has a perfectly flat meniscus. Steps to compensate and correct for this are time consuming and complex as it would require physically measuring the path length and adding complex calculations not normally achievable by the user.
For example, in vertical reading using a fixed vessel size, such as a microtiter well, volume is proportional to path length and path length is proportional to absorbance. Therefore, slight pipetting variations from vessel to vessel or lab to lab will have significant effects on the results. This would then require a widening of the acceptable ranges, thereby decreasing the sensitivity of the dye in detecting miscalibrated instruments.
The surface shapes of different liquids also effects path length, which in turn effects absorbance. For example, water can have either a flat surface or a sloping surface in the microtiter well. Since the user must read through a small aperture to avoid light distorting effects of the vessel, these two differently shaped surfaces would produce different path lengths, thus different absorbance readings.
Packaging of pre-calculated dyes in liquid form would not be feasible, due to leakage problems and the inability to restore the volume of the liquid to the microtiter well in order to obtain a reproducible result. If liquid dyes were pre-dispensed and distributed as liquids, it would be found that an amount of the liquid dye would tend to remain on the exposed sides of the microwell and any cover which is placed thereover. Some variable amount of the path length from which an original absorbance measurement was obtained would be lost, and the original absorbance value would thus be in error. Alternatively, liquid dyes which were not pre-dispensed would require pipetting precision and protection from evaporation of the stock solutions for accurate results.